Gene-to-metabolite network for biosynthesis of lignans in MeJA-elicited Isatis indigotica hairy root cultures.

Ruibing Chen,Qing Li,Ruifang Ma,Ying Xiao,Junfeng Chen,Philipp Zerbe,Hexin Tan,Shouhong Gao,Lei Zhang,Wansheng Chen

doi:10.3389/fpls.2015.00952

Abstract

Root and leaf tissue of Isatis indigotica shows notable anti-viral efficacy, and are widely used as “Banlangen” and “Daqingye” in traditional Chinese medicine. The plants' pharmacological activity is attributed to phenylpropanoids, especially a group of lignan metabolites. However, the biosynthesis of lignans in I. indigotica remains opaque. This study describes the discovery and analysis of biosynthetic genes and AP2/ERF-type transcription factors involved in lignan biosynthesis in I. indigotica. MeJA treatment revealed differential expression of three genes involved in phenylpropanoid backbone biosynthesis (IiPAL, IiC4H, Ii4CL), five genes involved in lignan biosynthesis (IiCAD, IiC3H, IiCCR, IiDIR, and IiPLR), and 112 putative AP2/ERF transcription factors. In addition, four intermediates of lariciresinol biosynthesis were found to be induced. Based on these results, a canonical correlation analysis using Pearson's correlation coefficient was performed to construct gene-to-metabolite networks and identify putative key genes and rate-limiting reactions in lignan biosynthesis. Over-expression of IiC3H, identified as a key pathway gene, was used for metabolic engineering of I. indigotica hairy roots, and resulted in an increase in lariciresinol production. These findings illustrate the utility of canonical correlation analysis for the discovery and metabolic engineering of key metabolic genes in plants.

Highlights

Isatis indigotica Fortune has been used in traditional Chinese medicine for more than two millennia and is listed in the Chinese Pharmacopoeia (National Pharmacopoeia Committee, 2010)
Identification of AP2/ERFs in the I. indigotica Transcriptome A total of 112 putative AP2/ERFs, designated Ii001 to Ii112, were obtained through query of a previously established I. indigotica transcriptome inventory (Chen et al, 2013) against public AP2/ERF and AP2/ERFs-like protein sequences of A. thaliana and B. rapa by TBLASTN (Basic Local Alignment Search Tool 2.2.26) (Table S3)
Yang and coworkers reported that AtERF073 (AT1G72360) modulated ethylene responses during hypoxia in A. thaliana (Yang et al, 2011)

Summary

Introduction

Isatis indigotica Fortune has been used in traditional Chinese medicine for more than two millennia and is listed in the Chinese Pharmacopoeia (National Pharmacopoeia Committee, 2010). Lignans including lariciresinol and larch lignan glycosides were considered as the material base of those activities (Yang et al, 2013). Lignan biosynthesis in Isatis indigotica analysis of I. indigotica (Chen et al, 2013) and availability of the complete genomes of other lignan-forming plant species (A. thaliana and Chinese cabbage) offer the opportunity to employ bioinformatics tools for better understanding and modulating lignan metabolism in I. indigotica. Key genes responsible for the biosynthesis of backbone structures of phenylpropanoids, flavonoids, lignans and lignins have been established, including phenylalanine-ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H) and coumaroyl-CoAligase (4CL) of phenylpropanoid metabolism, chalcone synthase (CHS), flavonol synthase (FNS) and chalcone isomerase (CHI) of flavonoid biosynthesis, and cinnamoyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) in lignan formation (Figure 1A)

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Conclusion